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Research ArticleBiochemical and Molecular Phylogenetic Study of AgriculturallyUseful Association of a Nitrogen-Fixing Cyanobacterium andNodule Sinorhizobium with Medicago sativa L.

E. V. Karaushu,1 I. V. Lazebnaya,2 T. R. Kravzova,3 N. A. Vorobey,4

O. E. Lazebny,5 D. A. Kiriziy,4 O. P. Olkhovich,1 N. Yu. Taran,1 S. Ya. Kots,4

A. A. Popova,6 E. Omarova,7 and O. A. Koksharova6,7

1Educational and Scientific “Institute of Biology”, Taras Shevchenko National University of Kyiv,64/13 Volodymyrska Street, Kyiv 01601, Ukraine2N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkin Street 3, Moscow 119333, Russia3Lomonosov Moscow State University, Biocenter, Leninskie Gory 1-12, Moscow 119991, Russia4Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine, 31/17 Vasylkivska Street, Kyiv 03022, Ukraine5N. K. Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, Vavilova Street 26, Moscow 119334, Russia6Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia7Lomonosov Moscow State University, Belozersky Institute of Physical-Chemical Biology, Leninskie Gory 1-40, Moscow 119992, Russia

Correspondence should be addressed to O. A. Koksharova; [email protected]

Received 11 September 2014; Accepted 24 February 2015

Academic Editor: Peter F. Stadler

Copyright © 2015 E. V. Karaushu et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standardnitrogen treatment methods. Alfalfa plants were inoculated bymono- and binary compositions of nitrogen-fixingmicroorganisms.Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium melilotiT17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. Thistreatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and anintensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis andleads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves.The full nucleotide sequenceof the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH) gene ofNostoc PTVwere deposited toGenBank(JQ259185.1, JQ259186.1). Comparison of these gene sequences ofNostoc PTVwith all sequences present at the GenBank shows thatthis cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showedthat this cyanobacterium clustered with high credibility values with Nostoc muscorum.

1. Introduction

Continuous anthropogenic impact on the environment ofdifferent chemicals, fertilizers, herbicides, plant protectionfrom pests and diseases, plant growth regulators, and so forththat are used in agriculture, makes it necessary to developan alternative to agricultural production, which would bebased on the use of cost-effective and environmentally

friendly systems for land application of fertilizers and plantprotection. An important role in this respect is given to themaximum use of the soil microflora.

In many countries around the world, studies and imple-mentation of the compositions consisting of symbiotic andfree-living nitrogen-fixing microorganisms have started toincrease productivity of crops. Among the wide range ofdiazotrophic microorganisms cyanobacteria are the most

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 202597, 16 pageshttp://dx.doi.org/10.1155/2015/202597

2 BioMed Research International

versatile for biochemical potential, since they do not need tobe provided with soil organic substances for nitrogen fixationunlike heterotrophic nitrogen-fixing microorganisms.

Positive ecological role of cyanobacteria in the soil asnitrogen-fixing bacteria which participate in deposition oforganic matter is well known now, and besides they arethe centers of microcosms as autotrophic organisms withamazing abilities for symbiotrophic relations [1, 2]. The lastproperty of cyanobacteria is particularly interesting in caseof using the consortia of microorganisms in biotechnologyinstead of monocultures [3]. In nature, cyanobacteria arenever found in the form of cell populations of one species.They are in a close relationship with the microbial commu-nity, located in the mucus of the surrounding cells. Researchin this field has shown that the composition of satellitecyanobacteria is very labile and it depends on changes inhabitat conditions. Axenic cultures of cyanobacteria existonly in the laboratories. In nature, they form a commu-nity and, being the edificators of microbial communities,cyanobacteria can change the microbial composition [4].It allows the constructing of artificial microbial consor-tium. Nitrogen-fixing activity (NFA) of soil compositionsof diazotrophic microorganisms can be an effective wayto supply the crop by environmentally friendly biologicalnitrogen. Use of this approach requires in-depth study of therelationship between bacteria, cyanobacteria, and plants, aswell as compatibility of microorganisms-partners in createdartificial associations. It is important to perform the screeningof the most suitable strains of microorganisms, to createconditions for the effective functioning of these symbioticconsortia. It is necessary to select an optimal quantitativeratio of microorganisms and methods of their implantationinto the rhizosphere.

The goals of this research were the study of the effectof artificial stable microbial consortium based on nitrogen-fixing cyanobacterium Nostoc PTV and Tn5-mutant of nod-ule bacteria Sinorhizobium meliloti T17, on the physiologicaland biochemical characteristics of growth and developmentof alfalfa, and, finally, on its yield and product quality andthe molecular typing and phylogenetic analysis of this newcyanobacterial isolate Nostoc PTV.

2. Material and Methods

2.1. Organisms and Growth Conditions. Plant alfalfa Med-icago sativa (L.) sort of Jaroslavna obtained from the NSCInstitute of Agriculture of National Academy of AgrarianSciences of Ukraine has been used in the experiments. Forthe inoculation of alfalfa seeds we used the strain of nodulebacteria Sinorhizobium (Rhizobium) meliloti T17 (patent ofUkraine number 55432) from the collection of nitrogen-fixing microorganisms of the Institute of Plant Physiologyand Genetics, National Academy of Sciences of Ukraine(Kyiv) [6]. The strain of S. meliloti T17 was obtained as aresult of intergeneric conjugation of Escherichia coli S17-1(pSUP2021::Tn5) and S. meliloti 425a on agar medium TY(tryptone/yeast extract) as described in [7] and it was selectedfor improved symbiotic properties. To create the binarycomposition of nitrogen-fixing microorganisms the culture

of cyanobacterium Nostoc PTV (from the collection of theInstitute of Hydrobiology, National Academy of Sciences ofUkraine) was used. Cyanobacteriumwas grown on Fitzgeraldmedium with the modification by Zehnder and Gorham [8]in Erlenmeyer flask at 22∘C ± 2∘C and illumination of 2500lux until the stationary growth phase. The concentration ofchlorophyll (Chl) in cyanobacterial cells was determined bydifferential fluorometry (Fluorometer FL300 3M, Russia) [9].The binary composition was prepared bymixing the bacterialsuspensions consisting of nodule bacteria (1 × 109 cells/mL)and cyanobacteria (Chl, mg/L = 1506,6 ± 13,4,Δ𝐹 = 0,088) inthe ratio 1 : 1. In parallel, the viability of cyanobacterial cellswas determined by the difference of fluorescence intensity(Δ𝐹) before and after the addition of simazine, the inhibitorof cells photosynthetic electron transport [10, 11].

Investigations were carried out in the model experimentsin a growth area of Institute of Plant Physiology and Geneticswith natural light and humidity of the substrate 60% of fullcapacity. Plastic containers with 10 kg of sand were used inexperiments. 12 alfalfa plants were grown in each container.Containers were preliminarily sterilized with 20% solution ofH2O2. Washed river sand with the mineral nutrient mixture

of Gelrigel [12] containing the “start” of nitrogen (177mg ofCa(NO

3)2× 4H2O per 1 kg of sand) was used as a substrate.

This amount of nitrogen represents one-quarter of the normalnitrogen supply. Before sowing the seeds were sterilized withconcentrated sulfuric acid for 5 minutes, and then they werewashed in running water for 1 h. The treatment of seeds bymicroorganism compositions was continuing during 1 h.

The controls in the experiments were the samples ofseeds treated by monoculture of T17 S. meliloti or only byN. PTV. We used samples of alfalfa seeds moisturized withtap water as an additional “absolute” control. Experimentswere performed in seven replications. Plants for analysis wereselected in phases of stem (32nd day of emergence), budding(40th day), and flowering (50th day).

2.2. Measurements of Nitrogen Activity, Pigments Content, andEfficiency of Photosynthesis. Nitrogen-fixing (nitrogenase)activity was determined by the level of activity of root nodulesby acetylenemethod and expressed asmicromoles of ethyleneformed by nodules per plant for 1 h [13]. The gas mixturewas analyzed by gas chromatography of Agilent Technologies6855 Network GC System (USA). The measurements wereperformed in five replications.

The content of the photosynthetic pigments in leavesof alfalfa plants was determined by the Wellburn method[14]. Pigments were extracted with dimethyl sulfoxide (0.1 gvegetable material was treated in 10mL DMSO) of leaf cutfor 3 h at +67∘C until complete extraction. The absorbanceof the solution was measured by spectrophotometer SmartSpec Plus (BioRad, USA) at 665 and 649 nm in a 1 cmcuvette. Leaves were collected from themiddle tiers of the fiverandomized plants of the same version. Measurements wereperformed in triplicate.

The net assimilation rate of shoots was determined incontrolled environment with installation built on base ofthe photoacoustic infrared gas analyzer GIAM-5M (Russia),

BioMed Research International 3

which was connected by differential circuit. Container withplants was placed in sealed plexiglass chamber of 50 litersthrough which air was blown at rate of 15 L/min. At the outletof chamber 1 L/min of air was taken to the gas analyzer, andthe remaining air was discharged into atmosphere.The cham-ber was irradiated with light by the lamp CG-2000 througha water filter. The illumination on the substrate level was250W/m2; temperature was 25∘ ± 2∘C. After the adaptationof plants to the conditions of measurement (30–40min afterclosing the chamber), the rate of absorption of CO

2by plants

was recorded (it is an apparent photosynthesis). After this,shoots of plants were cut at the substrate level and respirationof soil with roots were measured. Net assimilation rate wascalculated as sum of apparent photosynthesis and respiration.Calculations of gas exchange parameters were performedaccording to the standard procedure [15].

The protein content was determined in leaves of alfalfaplants in the budding stage by Lowrymethod [16]. Qualitativeand quantitative composition of amino acids was determinedby liquid-ion exchange column chromatography with the useof automatic analyzer T339 (Czech) on the basis of ninhydrindetection method [17].

2.3. Plant Stress Resistance Determination. In order to studythe effect of mono- and binary inoculation on plant resis-tance, the basic parameters of the stress state of alfalfa weredetermined. Plants in the budding stage were treated withherbicide diquat (100 pmol), which was used as a stress factor.Sampling was carried out after 30 minutes, 60 minutes, and24 hours of diquat action on plants. Specific changes in thecomposition of the components of a lipid-pigment complexand antioxidant systemwere studied in photosynthetic tissuesof alfalfa.

Intensity of lipid peroxidation (LPO) was evaluated bythe number of end-products of lipid oxidation based on thereaction with 2-thiobarbituric acid (TBA) [18]. The activityof antioxidant systems is determined by the activity ofsuperoxide dismutase (SOD) [19].

A statistical analysis of the experimental data was per-formed by standard methods, involving a package of specialstatistical functions of Microsoft Excel. Probability of dif-ferences between the variants was assessed by t-test and asignificance level of 𝑃 < 0,05.

2.4. Scanning Electron Microscopy (SEM). Cyanobacterialsamples were fixed as described above and dehydratedthrough an ethanol series, with an overnight exposure inabsolute acetone followed by critical-point drying in a DryerHCP-2 (Hitachi, Japan), coated with Au-Pd alloy in an IB-3Ion Coater (Eiko, Japan) and examined with a JSM-6380LAscanning electron microscope (JEOL, Japan).

2.5. DNA Isolation and PCR Amplification. For moleculartyping cyanobacterial genomic DNA was isolated accordingto [20] and synthetic oligonucleotides (“Synthol,” Moscow,Russia) have been used as cyanobacterial primers for 16S–23S rRNA PCR, according to [21]. As a second molecularmarker the nifH gene has been used with corresponding PCRprimers [22]. PCR for 16S–23S rRNA gene cluster was carried

out on a Tercik DNA amplifier (DNA Technology, Russia) byusing DreamTaq PCR Master Mix (Fermentas, EU), underthe following conditions: 1 cycle at 94∘C for 10min, 25 cyclesat 94∘C for 45 sec, 54∘C for 45 sec, 68∘C for 2min, 1 cycle at68∘C for 7min, and a final soak step at 4∘C. PCR for partialnifH gene was performed under the following conditions: 1cycle at 94∘C for 4min, 25 cycles at 94∘C for 30 sec, 54∘C for30 sec, 68∘C for 30 sec, 1 cycle at 68∘C for 7min, and a finalsoak step at 4∘C. PCR products were resolved in 1.5% agarosegel containing ethidium bromide at 5 microgram mL−1.

2.6. Cloning and Sequencing of PCR Products. DNA frag-ments obtained during PCR were cloned with CloneJet PCRCloning Kit # K1231 (Fermentas, EU). Transformation ofcompetent XL-1 cells of Escherichia coli and plasmid purifica-tion were performed according to [23]. DNA sequencing wasperformedwith ABI PRISMBigDye Terminator version 3.1 atthe Applied Biosystems 3730 DNAAnalyzer (Center for Col-lective Use “Genome”). Sequences were edited and assembledwith Bioedit (Invitrogen, Carlsbad, CA). The full nucleotidesequence of the rRNA gene cluster of cyanobacteriumNostocPTV and a part of the nifH gene were accomplished anddeposited to GenBank under accession numbers JQ259185.1and JQ259186.1.

2.7. Phylogenetic Analysis. Search of the nucleotide sequencesin the database GenBank, homologous to the sequencedgenes of studied species of cyanobacteria, was performedusing BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO-GRAM=blastn&PAGE TYPE=BlastSearch&LINK LOC=blasthome) with the option: the least degree of similarity(Minimum Identity). The sequences of selected specieswere aligned using the algorithm Muscle (MEGA 6.0) [24].Phylogenetic reconstructions were performed using Bayesianinference (MrBayes version 3.1.2) [25] with the preselectionof an adequate model of nucleotide substitutions (MEGA6.0).

2.8. Mating and Conjugal Transfer of Plasmid DNA. Trans-formations of Nostoc PTV through triparental conjugationsfollowed published protocols [26] with minor modifications.Standard bacterial mating involved the cyanobacterial strainNostoc PTV and E. coli strains (DH10B) that harbored thefollowing three plasmids: (i) the conjugal plasmid pRL443[27], (ii) the “helper” plasmid pRL623, [27], and (iii) thecargo plasmid pRL692 that carries the mobile element Tn5-692 [28]. E. coli strains were grown in 3mL LB with theappropriate antibiotic(s) and incubated at 37∘C overnight.Cells of E. coli were diluted 1 : 20 and were grown for 1.5–2 h at 37∘C. Then E. coli cells were harvested from 1mL ofeach E. coli culture by centrifugation and resuspended in1mL fresh LB. This step was repeated twice to wash the cells.After the third centrifugation, the cells were resuspendedin 200mL BG-11. Five milliliters of a growing Nostoc PTVculture was harvested by centrifugation at low speed (4000 g)and resuspended in 1mL BG-11. Then the filaments werefragmented in a water bath sonicator for 2 to 5min so thatmore than half of the filaments were shorter than 5 cells.


Table 1:Dynamics of accumulation of vegetativemass of alfalfa inoculated bymono- and binary suspensions of diazotrophicmicroorganisms.

Inoculants

Phase of plant developmentStooling Budding Flowering

Above-groundmass

(g/plant)

Mass of roots(g/plant)

Above-groundmass

(g/plant)


Above-groundmass

(g/plant)


Without inoculation(control) 0,42 ± 0,02 0,12 ± 0,01 1,17 ± 0,06 1,12 ± 0,1 1,25 ± 0,02 2,25 ± 0,16

N. PTV 0,62 ± 0,02 0,18 ± 0,01 1,64 ± 0,09 1,79 ± 0,09 1,70 ± 0,07 2,11 ± 0,15S. meliloti f17 0,65 ± 0,04 0,15 ± 0,01 1,59 ± 0,13 1,22 ± 0,11 1,83 ± 0,04 2,59 ± 0,24S. meliloti f17 + N.PTV 0,75 ± 0,08 0,22 ± 0,01 1,81 ± 0,18 1,82 ± 0,10 1,92 ± 0,03 2,95 ± 0,29

𝑃 ≤ 0,05.

The cyanobacterial cells were collected by centrifugation for2min and resuspended in 1mL BG-11. The cargo strain,the conjugal strain (for triparental mating), and NostocPTV were combined, pelleted by centrifugation, and finallyresuspended in 200mL BG-11. The conjugation mixture wasincubated for about 1 h in low light at 28∘C. Then the cellswere spread on sterile nitrocellulose filters laid on BG11+ 5%(vol/vol) LB agar plates (mating plates). The mating plateswere incubated without antibiotic selection for 18 to 24 h inlow light at 28∘C, and then the filterswere transferred toBG-11for 24 h and then to BG-11 agar with 10𝜇g/mL Spectinomycin(Sp10) and 2 𝜇g/mL Streptomycin (Sm2). After incubationfor 8 to 12 days, isolated antibiotic-resistant transconjugantcolonies were patched on fresh selective BG-11 plates.

3. Results and Discussion

3.1. Effect of Microbial Inoculation on Plant Growth andProductivity. Earlier in the laboratory study of pure culturesof N. PTV and S. meliloti [29], we found stimulation ofcell growth area of nodule bacteria around the colonies ofcyanobacterium N. PTV on the surface of the agar medium.Our results are consistent with the literature data, since itis known that cyanobacteria are producers of a wide rangeof biologically active substances, which include a group ofgrowth-stimulating compounds, analogues of phytohormone[30].

In our previous studywe have tested different associationsof nitrogen-fixing microorganisms in the rhizosphere ofalfalfa [29]. The most effective bacterial consortium includedcyanobacterium Nostoc PTV and Tn5-mutants of nodulebacteria S. meliloti.Usage of the optimal proportions of com-ponents in the inoculation mixtures promotes the absenceof antagonism between microorganisms and provides thestimulating effect of these consortia on various physiologicaland biochemical features of alfalfa plants.

In this study the possibility of the formation of artifi-cial stable microbial consortium based on nitrogen-fixingcyanobacterium N. PTV (Figure 1) and one Tn5-mutantof nodule bacteria S. meliloti T17 was investigated. In potexperiments it was revealed that inoculation of alfalfa bybinary mixture of S. meliloti T17 + N. PTV has a stimulatingeffect on the growth of the vegetative mass of plants (Table 1).

Figure 1: A SEM image of the N. PTV cells. Scale bar: 5𝜇m.

The increase of above-ground plant mass after applicationof the consortium S. meliloti T17 + N. PTV in the phaseof stem was 15.4% compared with rhizobial T17 monoinoc-ulation and 21% compared with N. PTV monoinoculation,respectively. The growth of above-ground alfalfa plant masswas 13.8% and 10.4%, after application of the S. melilotiT17 + N. PTV consortium in the budding stage, and 5%and 13% in the beginning of flowering, in comparisonwith monoinoculations, correspondingly. It is known thatcells of nitrogen-fixing cyanobacteria produce polypeptides,amino acids, polysaccharides, and vitamins. Due to thisdiverse biochemical activity in the mucous environmentof cyanobacterial cells favorable conditions for growth andreproduction of othermicroorganismswere created. Perhaps,it could promote a more active cell proliferation of nodulebacteria T17 associated with cyanobacteria in the root zoneof alfalfa and contribute to formation of efficient Rhizobium-legume symbiosis.

Rhizogenesis was positively affected in plants, the seedsof which were treated with suspensions of microorganisms(Table 1). The largest increase of the plant root mass wasdetected after using the binary inoculation (S. meliloti T17 +N. PTV). Thus, in the phase of stem the mass of rootsincreased by 46.6% and 22.2%, in the budding stage by 49.2%and 13%, and in the early phase of flowering by 13.9% and39.8%, compared with plants treated only by S. meliloti T17 orby only N. PTV, correspondingly.


Table 2: Number and mass of root nodules on alfalfa plants inoculated by mono- and binary suspensions of microorganisms.

Inoculants

Phase of plant developmentStooling Budding Flowering

Number of rootnodules

(pcs/plant)

Mass of rootnodules(g/plant)


(pcs/plant)



(pcs/plant)


Without inoculation(control) 0 0 0 0 0 0

N. PTV 0 0 0 0 0 0S. meliloti f17 12,0 ± 1,0 0,010 ± 0,00 30,0 ± 8,5 0,115 ± 0,002 45,0 ± 0,5 0,135 ± 0,02S. meliloti f17 + N.PTV 14,0 ± 0,6 0,017 ± 0,04 57,0 ± 8,0 0,130 ± 0,001 70,0 ± 7,5 0,160 ± 0,02

Note. 15 plants of each variant of the experiment were analyzed for determination the average number of nodules on the roots of one plant.𝑃 ≤ 0,05.

0

2

4

6

8

10

12

1 2 3

Tn5-mutant T17Tn5-mutant T17 + Nostoc PTV

Nitr

ogen

-fixi

ng ac

tivity

(mic

rom

oles

C 2H4/

(pla

nt∗

h))

Figure 2: Dynamics of NFA of nodules of alfalfa plants inoculatedby mono- and binary suspensions of microorganisms (micromolesof ethylene formed by nodules per plant per 1 h). 1: phase of stooling,2: phase of budding, and 3: phase of flowering, 𝑃 ≤ 0,05.

Effective collaboration between all partners of symbiosisprovides the activation of several metabolic processes and,above all, the fixation of atmospheric nitrogen. As a resultof improved plant nutrition, their productivity increased andthe quality of bioproducts improved.

In the phase of stemwhen the process of nitrogen fixationwas still inactive, differences in NFA of nodules of alfalfaplants inoculated by mono- or binary bacterial complexeswere not significant (Figure 2). However, in the budding stageand in the early flowering stage a nitrogen fixation in rootnodules of plants infected with a mixture of S. meliloti T17 +N. PTV was more intensive. Positive regulatory role ofcyanobacterium N. PTV is obvious according to the resultspresented in Table 2. Only in case of binary inoculation,plants demonstrate an increase in number and in weightof formed nodules (Table 2). Thus, the application of thismicrobial consortium provided increased NFA in nodules ofalfalfa at the budding stage and maintained its relatively high

level at the beginning of the early flowering stage (Figure 2).Therefore, this data proves that N. PTV has a stimulatingeffect on the functioning of root nodule bacteria S. melilotiT17.

It is known that the use of active strains of root nodulebacteria and their associations with other microorganismsaffect the formation and functioning of the photosyntheticcomplexes through the nitrogen status of a host plant. Pres-ence of nitrogen available to plants determines the efficiencyof symbiotic systems.

Mono- and binary suspensions inoculations of seedsshowed positive dynamics of accumulation of photosyntheticpigments in the leaves of alfalfa compared with the absolutecontrol (Figure 3). The most significant differences wereobserved in plants whose seeds had been inoculated withnitrogen-fixing consortium of microorganisms (chlorophyll𝑎 and chlorophyll 𝑏 increased by 114.6 and 82.9%) comparedwith the corresponding option of treatment only by strainT17. It is known that the content of chlorophyll in the leavesis directly proportional to the intensity of nitrogen fixationand depends on symbiotic properties of root nodule bacteria[31–33]. Increasing the number of pigments in the leaves ofalfalfa inoculated with binary bacterial suspension indicatesthe ability of N. PTV to enhance the functional activity ofrhizobia, which are directly interfaced with the intensity ofnitrogen fixation.

Available forms of nitrogen, such as mineral and sym-biotrophic, positively affect not only the formation of highgrade, but also functional state of the plant photosyntheticapparatus. The net assimilation rate also demonstrates theeffectiveness of the binary composition S. meliloti T17 + N.PTV. In particular, in the budding stage of these plants thenet assimilation rate exceeded 12.7%, while in the phase ofearly flowering it increased by 43.7% of the correspondingrate during inoculation only by T17 (Figure 3).

The net assimilation rate of plant leaves typically isclosely correlated with the content of nitrogen, and nitrogenis presented mainly in amino acids and proteins. Rubisco,the major cell photosynthetic enzyme of CO

2assimilation,

represents more than half of the soluble cell proteins in leaf.Obviously, the intensification of NFA in the binary composi-tionwas themain reason for the increase of plant assimilation


0

0.5

1

1.5

2

2.5

1 2 3 4C

onte

nt o

f chl

orop

hyll

(mg/

g of

ra

w m

ater

ial)

(I)

Chlorophyll aChlorophyll b

0

2

4

6

8

10

12

14

16

1

II(a)

2b2a0

5

10

15

20

25

30

1

II(b)

2b2a

Net

assim

ilatio

n ra

te (m

gCO

2/(

plan

t·h))

Net

assim

ilatio

n ra

te (m

gCO

2/(

plan

t·h))

Figure 3: (I): content of chlorophyll (mg/g of rawmaterial) in leaves of alfalfa inoculated bymono- and binary suspensions ofmicroorganismsS. meliloti f17 and N. PTV. 1: control (without inoculation); 2: inoculation by 𝑁. PTV; 3: inoculation by S. meliloti f17; 4: inoculation byconsortium of S. melilotif17 +N. PTV. II: net assimilation rate (mgb\

2

/( plant⋅hour)) of alfalfa inoculated bymono- and binary suspensionsof microorganisms S. meliloti f17 and N. PTV. 1: inoculation by N. PTV; 2a: inoculation by S. meliloti f17; 2b: inoculation by consortium ofS. meliloti f17 + N. PTV (II(a): phase of budding and II(b): phase of flowering).

rate. However, it is possible that more active symbioticapparatus, which is formed on the roots of plants throughbinary inoculation, enhanced “request” on assimilates by theroot system, thereby stimulating the photosynthetic activityof plant leaves. There is a gradient of transport forms ofcarbon, particularly sucrose, between roots and leaves in theconduction system and it accelerates the outflow of carbonfrom the leaves. This, in turn, eliminates restrictions byphotosynthesis products imposed on the feedback principleand further accelerates photosynthetic carbon assimilation.Thus, the efficient operation of the symbiotic apparatus ininoculated plants greatly stimulated the accumulation ofphotosynthetic pigments and increased the net assimilationrate. The accumulation of organic matter contributes to theformation of the plant biomass, because the basis of thebiological productivity of the plant organism, including thosecapable of symbiotic nitrogen fixation, is photosyntheticcarbon assimilation [31].

In consequence of artificial inoculation of alfalfa seedsby consortium of nitrogen-fixing microorganisms S. meliloti

T17 + N. PTV the yield of green mass of plants increased by17.9% and the protein content in the leaves increased by 12.0%compared to monoinoculation by strain T17 (Table 3).This isan evidence of the effective interaction of test organisms inthe cyano-Rhizobium associations and their positive impacton the growth and physiological characteristics of alfalfaplants (Table 3).

The amino acid composition is the main criterion ofthe biological value of proteins. An index of a total aminoacid composition of vegetative mass of the experimentalinoculated variants of alfalfa plants increased in comparisonto the control (without bacterial inoculation).Themaximumquantity of lysine, the most essential and deficient aminoacid in humans and animals, was recorded in leaves of alfalfa(Table 4). As a result of using binary inoculation a totalamino acid composition increased by 25.1%, compared withthe case of inoculation only by T17. In particular, a quantityof essential amino acids increased by 33.9%, and a quantityof nonessential amino acids increased by 17.7% (Figure 4).At the same time, an increase of the content of methionine,


Table 3: Productivity and protein content in leaves of alfalfa, inoculated by mono- and binary suspensions of microorganisms.

InoculantsHarvest of green mass of plant, g/vessel Protein content in the leaves

I mowing II mowing Total harvest % to monoinoculation byrhizobium

% tomonoinoculation by

rhizobiumControl 17,68 ± 0,51 19,70 ± 0,64 37,38 13,2N. PTV 21,40 ± 0,52 24,22 ± 0,76 45,62 14,6S. meliloti f17 21,81 ± 0,48 25,17 ± 0,30 46,98 18,32S. meliloti f17 + N. PTV 26,26 ± 0,37∗ 29,15 ± 0,17∗ 55,41 117,9 20,52 112,0

Table 4: Amino acid content in leaves of alfalfa, inoculated by mono- and binary suspensions of microorganisms.

Amino acid Content of amino acids (mg/100mgDW)Control N. punctiforme f17 f17 + N. punctiforme

Gamma-aminobutyric acid 0,065 0,088 0,085 0,123Lysine 0,386 0,802 0,459 0,610Histidine 0,094 0,301 0,171 0,232Arginine 0,297 0,905 0,439 0,585Asparagine 0,675 0,882 0,748 0,779Threonine 0,278 0,623 0,383 0,496Serine 0,321 0,698 0,413 0,533Glutamic acid 0,876 1,931 1,083 1,395Proline 0,376 0,610 0,419 0,548Glycine 0,423 0,790 0,470 0,537Alanine 0,479 0,895 0,581 0,644Cysteine 0,054 0,262 0,057 0,079Valine 0,245 0,651 0,301 0,423Methionine 0,111 0,298 0,149 0,200Isoleucine 0,175 0,439 0,233 0,273Leucine 0,523 1,234 0,685 0,938Tyrosine 0,201 0,534 0,285 0,253Phenylalanine 0,398 0,835 0,453 0,626Total 5,977 12,779 7,417 9,276

histidine, arginine, and tyrosine was observed, which arepresent in small quantities in plant leaves, and this is oneof the factors limiting the rate of biosynthesis of proteins,especially in generative organs. The results are a direct proofof the positive impact of cyanobacterial inoculation on thequality of agricultural products.

3.2. Stress Response of Plants Inoculated with Microbial Con-sortium. It is known that the plant productivity rate andresistance index are inversely dependent values. A positiveeffect of the cyanobacterial consortium T17 + N. PTV on theproductivity of alfalfa is shown in our study. It was logical tostudy the effect of the binary inoculation on plant resistanceto the adverse effects of certain environmental factors. It isan extremely important issue. We have used herbicide diquatas a model stress factor. For a short duration (30 minutes)of diquat treatment the content of TBA-reactive products inphotosynthetic tissues of plants that were inoculated withthe strain T17 was reduced by 18% compared to plantswithout inoculation (control 2). In the case when plants were

inoculated with the consortium S. meliloti T17 + N. PTV thecontent of TBA-reactive products remained at the level ofcontrol. In the experimentswithmore prolonged action of thestress factor (60min) the content of TBA-reactive productsin plants inoculated only by the strain T17 decreased by16.9% and in the case of binary inoculation the content ofTBA-reactive products decreased by 25%. It should be notedthat after 24 h of plants exposure with diquat, regardless ofthe inoculation agent used, reducing the amount of TBA-active products in photosynthetic tissues was not observedcompared with the control. At the same time, in the exper-iment with the use of the consortium of microorganisms adifference (15.4%) with the inoculation only by strain T17 wasmarked (Figure 5). At short-term action of diquat (30min)a rate of SOD activity in photosynthetic tissues of plantsinoculated with S. meliloti T17 + N. PTV was 2.5 timeshigher than in controls and by 42.5% in plants, inoculatedby strain T17. After herbicide treatment during 60 minutesa significant altering of SOD activity in inoculated plants(irrespective of whether a mono- or binary inoculation) was


0

1

2

3

4

5

6

7

8

1 2 3 4

EssentialNonessential

P ≤ 0.05

Tota

l con

tent

of a

min

o ac

ids (

mg/100

mg

dam

p)

Figure 4: Total content of essential and nonessential amino acidsin leaves of alfalfa grown after mono- and binary inoculation bycyanorhizobial compositions ofmicroorganisms: 1: control (withoutinoculation), 2: monoinoculation of alfalfa seeds by cyanobacteriumN. PTV, 3: inoculation of alfalfa seeds by Tn5-mutant strain of S.melilotiT17, and 4: binary inoculation of alfalfa seeds by Tn5-mutantstrain of S. meliloti T17 + N. PTV.

0

0.5

1

1.5

2

2.5

3

1 2 3 4 1 2 3 4 1 2 3 4

A CB

(Mic

rom

oles

of T

BA-a

ctiv

e pro

duct

s/g

of ra

w m

ater

ial)

Figure 5: Content of TBA-active products in alfalfa leaves afterherbicide diquat treatment: 0: for 30min, B: for 60min, andC: for 24 h. 1: control (without inoculation and herbicide diquattreatment); 2: control (without inoculation, with herbicide diquattreatment); 3: inoculation by Tn5-mutant of S. meliloti f17, withherbicide diquat treatment; 4: inoculation by Tn5-mutant of S.meliloti f17+ N. PTV, with herbicide diquat treatment.

not observed. However, in comparison to the control, thisdifference was significant; the enzyme activity was increasedby 54%. Under long-term stress (24 h) in plants inoculatedwith strain T17, SOD activity remained at the same level asfor short-term exposure. In plants, the seeds were treatedwith consortium S. meliloti T17 + N. PTV, for the sameconditions; this index decreased by 23.4% compared with

0

10

20

30

40

50

60

70

80

90

SOD

activ

ity (a

.u.)

1 2 3 4 1 2 3 4 1 2 3 4

A CB

P ≤ 0.05

Figure 6: SOD activity in alfalfa leaves after herbicide diquat treat-ment: 0: in 30min, B: in 60min, and C: in 24 h. 1: control (withoutinoculation and herbicide diquat treatment); 2: control (withoutinoculation, with herbicide diquat treatment); 3: inoculation byTn5-mutant of S. meliloti f17, with herbicide diquat treatment; 4:inoculation by f17 + N. PTV, with herbicide diquat treatment.

the control and 34.4%, in comparison with the plants inocu-lated by strain T17 (Figure 6).Thus, the plants inoculatedwithalgae-rhizobial composition proved to be more resistant tooxidative stress. It is possible due to the increased level ofNFAof their symbiotic system and thus the increase in the numberof available forms of nitrogen for alfalfa plants and thepossible participation of NO in the defense reactions. In theliterature, there are two hypotheses about the mechanisms ofNO action under conditions of stress. First, NOmay act as anantioxidant, directly linking to ROS, thereby protecting cellsfrom damaging their actions [34]. Secondly, NO can act asa signaling molecule that triggers a cascade of reactions thatlead to the expression of specific genes [35]. In their chemicaland physical properties small molecule, rapid metabolism,lack of charging, and high diffusion coefficient of NO are wellsuited for the role of intracellular signaling mediator of plantstress responses.

Thus, the inoculation of alfalfa seeds by a consortiumof nitrogen-fixing microorganisms S. meliloti T17 + N. PTVincreased the nitrogenase activity of root nodules, increasedthe net assimilation rate, and increased productivity andproduct quality, and also the stability of alfalfa plants underthe influence of oxidative stress induced by herbicides.

3.3. Molecular Typing and Phylogenetic Analysis of Cyanobac-terium Nostoc PTV. One of the main goals of this studywas the molecular typing and phylogenetic analysis of a newcyanobacterial isolate N. PTV originated from the Instituteof Hydrobiology of Academy of Science of Ukraine. As it wasshown above, this cyanobacterium is effective for soil algal-ization. As a component of algae-rhizobium compositions,this cyanobacterium stimulates germinative energy, growth,and productivity of legumes.

To identify and to determine the phylogenetic positionsof the new cyanobacterial isolate N. PTV we used a partial


sequence of the nifH gene (342 bp), encoding nitrogenasereductase, and 16S ribosomal RNA gene cluster (1765 bp) asmolecular markers. Comparison of the nifH gene sequenceand rRNA gene cluster sequence of cyanobacterium N. PTVwith all the sequences present at the GenBank by using theprogramBlast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO-GRAM=blastn&PAGE TYPE=BlastSearch&LINK LOC=blasthome) shows that this strain has no full similarity withany of early investigated organisms.

Comparison of rRNA gene cluster sequence (1765 bp)of the cyanobacterium Nostoc sp. PTV revealed that thiscyanobacterium shows the highest similarity with severalstrains ofN.muscorum andN. commune (Table 5). In general,support for branching in the tree, based on a fragment of 16SrRNA gene sequence (Figure 7), is worse in comparison withthe reconstruction of the phylogeny of cyanobacteria basedon the sequence of the gene nifH (Figure 8).

The group of Nostoc strains and species, which includesNostoc PTV, forms a cluster with the minimum of allowablesupport, 0.95. Hierarchy of strains (HA 4355-MV2, PTV,and 8964) and Nostoc species (N. muscorum and N. linckia)cannot be evaluated because of the low topological supportof this site of the tree: credibility values range from 0.56 to0.94. It is difficult to discuss a relation ofNostoc PTV strain tostrain HA 4355-MV2, due to the very low values of the otherbranches in the cluster, and very scarce information aboutthe HA-MV2 4355 strain clearly does not help to solve theproblem of their possible relationship.

N. muscorum is discussed below.N. linckia is also a fresh-water strain and could be isolated in some terrestrial niches.Interestingly, the Nostoc strain UAM 307 is quite clearlydifferentiated from the other representatives of Nostocaceae(0.95). Being sufficiently close to strain of Nostoc PTV, thiscyanobacterium has some significant features that providedthe separation of this strain into the other branch of thecommon with Nostoc PTV cluster.

Even more interesting detail is that the last significantbranch (credibility value is equal to unity) of the clusteris formed by the strain of N. muscorum Ind33, which issignificantly aside not only from the desired strain of PTV,but also from the different strains of the same species (CCAP1453-22). Thus, the strain of Nostoc PTV has teamed up withmembers of their own genus.

Outgroup of this dendrogram is represented by twostrains of Rivularia (this kind of cyanobacteria forms het-eropolar threads; their trichomes are densely agglomerated,covered with a total mucus).The genus is represented only bythe species often living on calcareous substrates, but there arerare epilithic and epiphytic species.

Comparison of nifH sequence of cyanobacterium PTVrevealed that this strain shows the highest similarity withseveral strains of Nostoc (Table 6). The closest relative isNostoc muscorum UTAD N213, purified from rice paddy inMondego River Basin (Portugal). Phylogenetic analysis (Fig-ure 8) revealed that the cyanobacterium PTV forms a mini-cluster with Nostoc muscorum UTAD N213. N. muscorumis a free-living filamentous cyanobacterium, which inhabitsboth terrestrial and freshwater aquatic environments. They

are phototrophic organisms performing photosynthesis andalso fixing atmospheric nitrogen [36].

N. muscorum is the most common type of Nostoc interrestrial ecosystems and is widely spread, due to theadaptability to many adverse conditions. It forms a symbioticrelationship with many types of terrestrial plants and fungi.

It is known that N. muscorum has great effect on soilbiology and productivity which makes it an attractive soilinoculant. This cyanobacterium is able to obtain carbonand nitrogen from the air and has an advantage overheterotrophic soil inoculants, which are usually limited bycarbon [37].

It also benefits plants and other soil bacteria by increasingsoil organic matter in the form of carbohydrates and providesbiological organic nitrogen that can be assimilated by plants[38].N.muscorumhelps to create the environment conditionsto further colonization and growth by plants and othermicroorganisms [39].

Inoculation of the N. muscorum isolates caused asignificant effect on growth of wheat and maize plants.Cyanobacterial inoculation positively affected pigment con-tent, increased plant shoot and root dry weight, and increasedleaf area [40].

In general, the topology of the dendrogram (Figure 8) hasa good support; the main branch nodes are characterized byhigh credibility values.

Nostoc PTV is in the same cluster with Nostoc sp. UAM-362 and Nostoc commune. Nostoc sp. UAM-362 was isolatedfrom the rock surface of calcareous river with brackish waterin Spain: Muga, Girona (Northeast Spain). N. commune is acolonial species of cyanobacterium.Aswell asN. punctiforme,N. commune is able to survive in extreme conditions such aspolar regions and arid areas.

Three more clusters are presented as the parts of the largeone: two single clusters, represented by Nostoc sp. Baikal(nitrogen-fixing cyanobacteria from Lake Baikal) and byNodularia spumigena from family Nostocaceae. Nodulariaoccurs mainly in brackish or saline waters. Nodularia cellsoccasionally can form heavy algal blooms. Some strainsproduce a toxin (nodularin), which is harmful to humanhealth [41].

The third cluster is formed by four strains of Tolypothrixand by one strain of Nostoc sp. UAM-367 (isolated fromrock surface of calcareous river with brackish water in Spain:Muga, Girona). Cyanobacteria Tolypothrix grow in unpol-luted waters; several species are found in swamps, knownaerophilic species growing on the bark of trees, in the wetsands, on wet rocks, and so forth.

Two species, and one strain (PCC 7120) of Anabaena,representing a family of filamentous cyanobacteria Nosto-caceae, belong to this large cluster with a poor resolution (acredibility value of branching is 0.71). These cyanobacteriaexist in the form of plankton; some species are symbionts ofplants. Anabaena is one of the four genera of cyanobacteriathat produce neurotoxins. Anabaena is a model for the studyof cell differentiation and differential gene expression duringnitrogen fixation [42]. A. siamensis and A. sphaerica arefreshwater species. This mega cluster consisting of describedspecies of cyanobacteria is well differentiated from the other

10 BioMed Research InternationalTa

ble5:BL

AST

results

obtained

byqu

erying

the16S–23S

rRNAgene

cluste

rofN

ostocsp.PT

Vwith

GenBa

nkandgeograph

icalandecologicaloriginso

fthe

hits.

ClosestG

enBa

nkrelative

GenBa

nkaccessnu

mber

Query

coverage,%

Score,%

Identity,%𝐸value

Orig

inof

thes

trainandreference

Nosto

csp.HA4355-M

V2clo

nep9

DHQ847576

982872

970.0

ManiniholoCavew

all,near

Haena

USA

:Kauai,H

awaii

Nosto

cmuscorum

CCAP1453/22

HF6

78509

982531

930.0

ScottishAssociatio

nforM

arineS

cience,M

olecular

and

MicrobialBiolog

y,Dun

staffn

ageM

arineL

aboratory,

Oban,

PA37

1QA,U

nitedKingdo

m

Anabaena

varia

bilis

ATCC

29413

CP00

0117

982457

920.0

Ithasb

eenstu

died

extensively

foro

ver4

0yearsa

ndis

thes

trainof

choice

form

anylabo

ratorie

sthrou

ghou

tthew

orld

Nosto

csp.PC

C7120

BA00

0019

982453

920.0

Com

pleteg

enom

icsequ

ence

ofthefi

lamentous

nitro

gen-fix

ingcyanob

acteriu

mAn

abaena

sp.(No

stoc)

strainPC

C7120

isavailable

Nosto

ccom

mun

eNC1

clone

10EU

586726

982441

910.0

John

CarrollUniversity,20700,N

orth

Park

Boulevard,

University

Heights,

OH44

118,U

SA

Nosto

ccom

mun

eNC1

clone

M2

EU586725

982441

910.0

John


orth

Park

Boulevard,

University

Heights,

OH44

118,U

SA

Nosto

ccom

mun

eNC5

clone

10EU

586727

982437

910.0

Biolog

y,John


orth

Park

Boulevard,University

Heights,

OH44

118,U

SA

Nosto

ccom

mun

eNC1

EU784149

982428

910.0

Nostocc

ommun

eNC1

was

isolated

from

soil

(sub

aeroph

yt)inTrebon

/Czech

repu

blicin

2006.

Nosto

ccom

mun

eNC5

clone

11EU

586728

982423

910.0

Biolog

y,John


orth

Park

Boulevard,University

Heights,

OH44

118,U

SA

Nosto

cellipsosporum

CCAP1453/15

HE9

75023

942399

920.0

ScottishAssociatio

nforM

arineS

cience,M

olecular

and

MicrobialBiolog

y,Dun

staffn

ageM

arineL

aboratory,

ObanPA

371Q

A,U

nitedKingdo

m

Nosto

ccf.punctiformeB

ashk

irclo

ne6A

EU586732

962378

920.0

John


orth

Park

Boulevard,

University

Heights,

OH44

118,U

SA

Calothrix

sp.H

A4356-M

V2clo

nep8i

JN385289

982361

900.0

Cave

wallscraping,ManiniholoCa

venear

Haena,U

SA:

Kauai,Haw

aii

Calothrix

sp.H

A4340

LM2

KF417425

982361

900.0

Cave,U

SA:K

auai,H

awaii,ManiniholoCa

ve

Anabaena

sp.C

CAP1403/4A

HE9

75015

932360

920.0

ScottishAssociatio

nforM

arineS

cience,M

olecular

and

MicrobialBiolog

y,Dun

staffn

ageM

arineL

aboratory,

ObanPA

371Q

A,U

nitedKingdo

mNo

stocm

uscorum

CCAP1453/8

HF6

78508

932358

920.0

UnitedKingdo

m:Scotland

Calothrix

sp.H

A4356-M

V2clo

nep8i

JN385289

982356

900.0

Cave

wallscraping,ManiniholoCa

venear

Haena,U

SA:

Kauai,Haw

aii

Cylin

drosperm

ummoravicu

mCC

ALA

993

clone

operon

1KF

052607

972331

940.0

Cave

sediment,Cz

echRe

public:A

materskaC

ave,

SouthMoravia

Nosto

cpun

ctiform

ePCC

73102

CP001037

972331

910.0

Asymbion

tfrom

acycad

Nosto

csp.Peltigera

malacea

DB3

992

cyanob

iont

JX2194

8397

2322

910.0

Cyanob

iont

oflichenizedfung

iPeltigeramalacea,

Iceland

Nosto

cmuscorum

CCAP1453/20

HF6

78506

952318

910.0

ScottishAssociatio

nforM

arineS

cience,M

olecular

and

MicrobialBiolog

y,Dun

staffn

ageM

arineL

aboratory,

ObanPA

371Q

A,U

nitedKingdo

m

BioMed Research International 11Ta

ble5:Con

tinued.

ClosestG

enBa

nkrelative

GenBa

nkaccessnu

mber

Query

coverage,%

Score,%

Identity,%𝐸value

Orig

inof

thes

trainandreference

Nosto

csp.10Dp6

6EJQ

259187

982318

900.0

From

associationwith

Dynam

enapumila

L.,W

hiteSea,

Russia

Cylin

drosperm

umcatenatum

CCALA

999

clone

operon

1KF

052615

972309

940.0

Soil,Slovakia:forestabo

veStaraB

rzotinskaC

ave,

Slovak

Karst

Trich

ormus

anom

alus

HA4352

LM2

KF417426

962309

920.0

Cave,U

SA:K

auai,H

awaii,ManiniholoCa

veCy

lindrosperm

umsp.H

A4236-M

V2clo

nep4

JN385290

982309

890.0

Taro

field,M

akikiN

atureC

enter,USA

:Oahu,Haw

aii

Cylin

drosperm

umcatenatum

CCALA

996

clone

operon

1KF

052611

972307

940.0

Soil,Cz

echRe

public:A

materskaC

ave,SouthMoravia

Tolypothrix

campylonemoidesF

I5-M

K38

clone

p10D

JQ083654

982305

900.0

Sand

,USA

:FortIrw

inNTC

,San

Bernardino

Co.,

California

Cylin

drosperm

umcatenatum

CCALA

990

clone

operon

1KF

052601

952302

940.0

Soil,Cz

echRe

public:B

enesov

nadCe

rnou

,Sou

thBo

hemia

Spirirestisrafaelen

sisWJT-71-N

PBG6clo

nep1B

JQ083656

982300

900.0

Joshua

Tree

NationalP

ark,USA

:Joshu

aTreeF

orest,

SanBe

rnardino

Co.,C

alifo

rnia

Cylin

drosperm

umbadium

CCALA

1000

clone

operon

1KF

052616

972298

940.0

Recla

imed

coalmines

oil,USA

:Pyram

idState

Recreatio

nArea,Illinois

Cylin

drosperm

umcatenatum

CCALA

991

clone

operon

1KF

052603

972293

940.0

Soil,Cz

echRe

public:M

ostR

egion,

North

Bohemia

Spirirestisrafaelen

sisWJT-71-N

PBG6clo

nep1A

JQ083655

982291

890.0

Joshua

Tree

NationalP

ark,USA

:Joshu

aTreeF

orest,

SanBe

rnardino

Co.,C

alifo

rnia

Nosto

csp.Peltigera

mem

branacea

cyanob

iont

N6

JX975209

972289

910.0

Symbion

tofP

eltigeramem

branacea

lichen,

Iceland

Cylin

drosperm

umpellu

cidum

CCALA

992

clone

operon

1KF

052605

972287

940.0

Cave

sediment,Slovakia:D

lhac

hodb

ainDom

icaC

ave

syste

m,SlovakKa

rst

Hassalliasp.E

M2-HA1clone

p4B

HQ847555

982282

890.0

Soil,MojaveN

ationalP

reserve,USA

:San

Bernardino

Co.,C

alifo

rnia

Tolypothrix

tenu

isf.terrestris

UFS

-BI-NPM

V-1A

2-F0

6clo

nep13E

JQ083651

982277

890.0

Arid

soilaft

erab

urn,

foothills

oftheO

naqu

eeMts.

USA

:Utah

Nosto

ccf.commun

e257-16

HQ877826

962271

900.0

Subaerial,on

Bonampak’s

archeologicalbuildingwalls,

Mexico:Ch

iapas

Hassalliasp.C

NP3

-B3-C0

4clo

nep5D

HQ847556

982271

890.0

Soil,Needles

Distric

t,VirginiaPark,C

anyonlands

NationalP

ark,USA

:San

Bernardino

Co.,C

alifo

rnia

Unculturedcyanob

acteriu

mclo

neEm

ix3.12

JX887892

922269

910.0

Freshw

ater

microbialmat

Konstanz,G

ermany

Cylin

drosperm

ummuscic

olaSA

G44

.79

clone

operon

1KF

111150

962268

930.0

Soil

France:G

if-Sur-Yvette,Ile-de-France

Region

Tolypothrix

tenu

isf.terrestris

UFS

-BI-NPM

V-1A

2-F0

6clo

nep13F

JQ083652

982268

890.0

Arid

soilaft

erab

urn,

foothills

oftheO

naqu

eeMts.

USA

:Utah

Hassalliasp.C

M1-H

A11clo

nep8A

JQ083650

982268

890.0

Sand

yloam

near

gypsum

mine,

USA

:Clark

Mou

ntains,San

Bernardino

Co.,

California


Table5:Con

tinued.

ClosestG

enBa

nkrelative

GenBa

nkaccessnu

mber

Query

coverage,%

Score,%

Identity,%𝐸value

Orig

inof

thes

trainandreference

Hassalliasp.C

M1-H

A08

clone

p7B

JQ083648

982268

890.0

Sand

yloam

near

gypsum

mine,

USA

:Clark

Mou

ntains,San

Bernardino

Co.,

California

Nosto

ccf.commun

e257-20

HQ877827

942266

910.0

Biofi

lmso

fN.cf.commun

ewerec

ollected

atBo

nampakarcheologicalareain2008

from

twosites

ontheb

uildingwalls(C

hiapas,M

exico).

Tolypothrix

campylonemoidesF

I5-M

K38

clone

p10A

JQ083653

982266

890.0

Sand

USA

:FortIrw

inNTC

,San

Bernardino

Co.,

California

Hassalliasp.C

M1-H

A08

clone

p7F

HQ847554

982266

890.0

Soil,ClarkMou

ntains,n

earg

ypsum

mine

USA

:San

Bernardino

Co.,C

alifo

rnia

Desmonostocsp.HA7617

LM4

KF417429

962241

900.0

USA

:Kauai,H

awaii,WaikapalaeC

ave

Camptylonem

opsis

sp.H

A4241-M

V5clo

neB2

-3+p4

JN385292

962223

930.0

Molekas

tream

USA

:Oahu,Haw

aii

Anabaena

circin

alis33-10iso

late

EF6344

7498

2199

880.0

OhauCh

annel,New

Zealand

Tolypothrix

sp.P

CC7504

isolateDBS

U18

FJ66

0999

902194

920.0

Freshw

ater

Aquariu

m,Sweden

Nosto

csp.UA

M307

HM623782

702111

980.0

Rock

surfa

ceof

calcareous

river

Spain:

MatarranyaR

iver,Teruel,Ea

stSpain

Rivularia

sp.1PA

23FJ66

0980

922021

880.0

PozasA

zulesI,M

exico

Microbialite

freshwater

Nosto

csp.8964

:3AM711541

662006

990.0

HostisG

unneraprorepens(Ang

iospermae),New

Zealand

Rivularia

sp.1PA

21FJ66

0978

922004

880.0

Instituteof

Ecolog

y,UNAM

(Mexico)

Nosto

clinckiavar.arvenseIAM

M-30

AB3

25907

651999

990.0

Cultivatedsamples

from

theInstituteo

fMolecular

Biosciencesa

tthe

University

ofTo

kyo

Aphanizomenon

ovalisp

orum

ILC-

164

JF768745

921988

930.0

Lake

Kinn

eret,Israel;Ba

nker

etal.,1997

[5]

Nosto

cmuscorum

Ind33

HM573462

651988

990.0

Padd

yfield,

India:AgriculturalFarms,Ba

narasH

indu

University,

Varanasi,

Utta

rPradesh


Hassallia sp. EM2-HA1 clone p4B HQ847555Hassallia sp. CM1-HA08 clone p7B JQ083648Hassallia sp. CM1-HA11 clone p8A JQ083650

Tolypothrix tenuis f. terrestris UFS-BI-NPMV-1A2-F06 clone p13E JQ083651

Tolypothrix campylonemoides FI5-MK38 clone p10A JQ083653Tolypothrix campylonemoides FI5-MK38 clone p10D JQ083653

Spirirestis rafaelensis WJT-71-NPBG6 clone p1B JQ083656Spirirestis rafaelensis WJT-71-NPBG6 clone p1A JQ083655

12

2- Cylindrospermum sp. HA4236-MV2 clone p4 JN3852901- Cylindrospermum muscicola HA4236-MV2 JN385290

Anabaena circinalis 33-10 isolate EF634474Aphanizomenon ovalisporum ILC-164 JF768745.1

Calothrix sp. HA4356-MV2 clone p8i JN385289Calothrix sp. HA4340 LM2 KF417425Nostoc sp. 10Dp66E JQ259187

Nostoc commune NC1 clone M2 EU586725Nostoc commune NC1 clone 10 EU586726Nostoc commune NC1 EU784149Nostoc commune NC5 clone 10 EU586727Nostoc commune NC5 clone 11 EU5867283

45

6

3- Cylindrospermum catenatum CCALA 999 clone operon 1 KF0526154- Cylindrospermum catenatum CCALA 996 clone operon 1 KF052615- Cylindrospermum catenatum CCALA 990 clone operon 1 KF0526016- Cylindrospermum catenatum CCALA 991 clone operon 1 KF052603

Cylindrospermum muscicola SAG 44.79 clone operon 1 KF111150

Cylindrospermum badium CCALA 1000 clone operon 1 KF052616

100

95

86

100

100

58

100

100

9852

100 100

10078

83

66

86

90

98

100100

65

7284 100

97

Nostoc cf. commune 257-16 HQ877826Nostoc cf. commune 257-20 HQ877827

Nostoc sp. Peltigera malacea DB3992 cyanobiont JX219483Nostoc cf. punctiforme Bashkir clone 6A EU586732

Nostoc sp. Peltigera membranacea cyanobiont N6 JX975209Nostoc sp. Peltigera membranacea cyanobiont N6 JX975209

Uncultured cyanobacterium clone Emix3.12 JX887892

100100

77

86

97 100

10092

8- Nostoc ellipsosporum CCAP 1453 15 HE975023

95 6194

5685100 Rivularia sp. 1PA23 FJ660980

Rivularia sp. 1PA21 FJ660978

Nostoc sp. 8964 3 AM711541Nostoc linckia var. arvense IAM M-30 AB325907Nostoc muscorum CCAP 1453 22 HF678509Nostoc sp. PTV 16S JQ259185Nostoc sp. HA4355-MV2 clone p9D HQ847576

Nostoc sp. UAM 307 HM623782Nostoc muscorum Ind33 HM573462

Trichormus anomalus HA4352 LM2 KF417426Tolypothrix sp. PCC 7504 isolate DBSU 18 FJ660999

Desmonostoc sp. HA7617 LM4 KF417429Nostoc punctiforme PCC 73102 CP001037

Nostoc muscorum CCAP 1453 20 HF678506Anabaena variabilis ATCC 29413 CP000117

Nostoc muscorum CCAP 1453 8 partial HF678508Anabaena sp. CCAP 1403 4A HE975015Nostoc sp. PCC 7120 BA000019

8

7- Nostoc punctiforme PCC 73102 CP0010377

Cylindrospermum pellucidum_CCALA 992 clone operon 1 KF052605

Hassallia sp. CM1-HA08_clone p7F_HQ847554

Tolypothrix tenuis f. terrestris UFS-BI-NPMV-1A2F06clone_p13F JQ083652

Cylindrospermum moravicum CCALA993clone operon1KF052607

Figure 7: Phylogenetic relationships of Nostoc sp. PTV (designated by black square) inferred under the posterior probability criterion(MrBayes) from the gene for 16S rRNA, partial sequence information. Numbers at the nodes indicate the Bayesian statistical support values(posterior probabilities multiplied by 100); only values higher than 50% are given. The scale bar indicates the number of substitutions pernucleotide position.

Anabaena azollae L34879Anabaena variabilis U89346Nostoc PCC 6720 Z31716

Anabaena PCC7120 J05111Anabaena sp. A2 AF124377

Anabaena sp. L-31 L04499Anabaena PCC7120 AF012326

Anabaena siamensis TISTR 8012 DQ176436Anabaena sphaerica UTEX B 1616 DQ439648

Nostoc sp. Baikal JN887856Nodularia spumigena AV1 GQ456132

Tolypothrix sp. UAM 379 JQ514114Tolypothrix sp. UAM 415 JQ514120Tolypothrix sp. UAM 413 JQ514119

Tolypothrix sp. UAM 378 JQ514113Nostoc sp. UAM 367 JQ514116

Nostoc commune UTEX 584 L23514Nostoc sp. UAM 362 JQ514115

Nostoc sp. PTV JQ259186Nostoc muscorum UTAD N213 GQ443450

Mastigocladus laminosus CCMEE 5324 EF570558Fischerella UTEX1931 U49514

100

5885

98

100

7161

100

100

100

100

10098

78

93

0.05

Figure 8: Phylogenetic relationships of Nostoc sp. PTV (designated by black square) inferred under the posterior probability criterion(MrBayes) from the gene for nifH, partial sequence information. Numbers at the nodes indicate the Bayesian statistical support values(posterior probabilities multiplied by 100); only values higher than 50% are given. The scale bar indicates the number of substitutions pernucleotide position.


Table6:BL

AST

results

obtained

byqu

erying

then

ifHgene

ofNo

stocsp.PT

Vwith

GenBa

nkandgeograph

icalandecologicaloriginso

fthe

hits.

ClosestG

enBa

nkrelativ

eGenBa

nknu

mber

Query

coverage

%Score%

Identity%𝐸value

Orig

inof

thes

trainandreference

Nosto

cmuscorum

UTA

DN213

GQ443450.1

100

612

997𝑒−172

Rice

padd

yin

Mon

dego

RiverB

asin,Portugal

Nosto

cmuscorum

clone

CC1090A1

AY221814.1

94576

995𝑒−161

Ocean

Sciences

Departm

ent,University

ofCa

lifornia,SantaC

ruz,CA

9506

4,USA

Nosto

ccom

mun

e(UTE

X584)

L23514.1

99565

979𝐸−158

Scotland

Nosto

csp.UA

M362

JQ514115.1

99553

965𝑒−154

Rock

surfa

ceof

calcareous

river

with

brackish

water,

Spain:

Muga,Giro

na(N

ortheastSpain)

Nosto

csp.Ba

ikal

JN887856.1

99517

944𝑒−143

Nitrogen-fixing

cyanob

acteria

from

Lake

Baikal

Nodu

laria

spum

igenaAV

1GQ456132.1

99511

932𝑒−141

Thes

urface

waterso

fthe

BalticS

ea,Stockho

lm,Sweden

Tolypothrix

sp.U

AM

379

JQ514114.1

99462

908𝐸−127

Rock

surfa

ceof

calcareous

river

with

brackish

water,

Spain:

Muga,Giro

na(N

ortheastSpain)

Tolypothrix

sp.U

AM

378

JQ514113.1

99462

908𝐸−127

Rock

surfa

ceof

calcareous

river

with

brackish

water,

Spain:

Muga,Giro

na(N

ortheastSpain)

Tolypothrix

sp.U

AM

415

JQ514120.1

99453

894𝐸−124

Rock

surfa

ceof

calcareous

river

with

brackish

water,

Spain:

Muga,Giro

na(N

ortheastSpain)

Tolypothrix

sp.U

AM

413

JQ514119.1

99453

894𝐸−124

Rock

surfa

ceof

calcareous

river

with

brackish

water,

Spain:

Muga,Giro

na(N

ortheastSpain)

Nosto

cPCC

6720

Z31716.1

99430

884𝐸−117

Nosto

cPCC

6720

was

previouslykn

ownas

Anabaenopsiscircularis.

Thisisafreshwater

species

Anabaena

sp.L-31

L044

99.1

99426

885𝐸−116

Thefi

lamentous,heterocystous,n

itrogen-fixing

freshwater

cyanob

acteriu

mAn

abaena

PCC7

120

J05111.1

99426

885𝐸−116

http://wiki.ann

otation.jp/Kazusa:C

yano

Base:Anabaenasp.P

CC7120

Nosto

csp.UA

M367

JQ514116.1

100

423

877𝐸−115

Rock

surfa

ceof

calcareous

river

with

brackish

water,

Spain:

Muga,Giro

na(N

ortheastSpain)

Anabaena

siamensis

TIST

R8012

DQ176436.2

100

414

873𝐸−112

Anabaena

siamensis

isafi

lamentous

heterocysto

usnitro

gen-fix

ing

cyanob

acteriu

mwhich

originallywas

isolatedfro

mar

icep

addy

field

inTh

ailand

Mastigocladu

slam

inosus

CCMEE

5324

EF570558.1

100

414

873𝐸−112

Thec

osmop

olitanthermop

hilic

cyanob

acteriu

mMastigocladu

slaminosus

from

theU

niversity

ofOregon’s

Cultu

reCollectionof

Microorganism

sfrom

Extre

meE

nviro

nments(C

CMEE

)Fischerella

UTE

X1931

U49514.1

100

414

873𝐸−112

Thermop

hilic

cyanob

acteriu

m(syn

onym

:Mastigocladu

slam

inosus)

Anabaena

sphaerica

UTE

X“B

1616”

DQ4396

48.1

9940

887

1𝐸−110

Departm

ento

fChemistry

andCh

emicalEn

gineering,University

ofSheffi

eld,MappinStreet,Sheffield,

SouthYo

rkshire

S13JD,U

nitedKingdo

m

Anabaena

sp.A

2AF124377.1

9940

887

1.𝐸−110

Molecular

Evolution,

BMC,

Upp

salaUniversity,H

usargatan3,7512

4Upp

sala,Sweden

Anabaena

azollae

L34879.1

99435

881𝐸−118

Anabaena

azollae1a,ap

utatives

ymbion

tofA

zolla

carolin

iana

Anabaena

varia

bilis

U89346.1

99435

881𝐸−118

Anabaena

varia

bilis

ATCC

29413isafi

lamentous

cyanob

acteriu

mthatprod

uces

heterocysts

andfixes

nitro

genun

dera

varie

tyof

environm

entalcon

ditio

ns


two clusters: a single one represented by strain Anabaena L-31 (freshwater cyanobacterium) and a poorly differentiatedcluster, which consists of two strains of Anabaena (A2 andPCC 7120) and two well-differentiated species of Anabaena,A. azollae and A. variabilis. A. azollae forms symbiosis withwater fern Azolla.

Outgroup for the described species of Nostoc andAnabaena is represented by Mastigocladus laminosus and bymember of the genus Fischerella (strain UTEX 1931).The firstorganism is a typical representative of the genus Mastigo-cladus. Fischerella represents another squad, Stigonematales.Both types of reference are truly branching filamentous formsof thermophilic cyanobacteria.

3.4. Gene Transfer into Nostoc PTV Cells. Nostoc PTV cellswere tested for their ability to conjugational DNA transfer.As a result of plasmid pRL692 transfer into cyanobacterialcells several hundred transconjugant colonies were selectedon selective plates that contained solid BG-11 medium andantibiotics (Sp10 and Sm2). On control plates antibiotic-resistant colonies were absent (data not shown). In futureexperiments we plan to use this experimental approach fortransposon mutagenesis of Nostoc PTV and selection of thenew mutants with interesting characteristics.

4. Conclusions

Theuse ofmicrobial consortiumnewly identifiedNostocPTVstrain together with Sinorhizobium meliloti T17 was moreefficient than the use of the single-rhizobium strain for alfalfaplant inoculation. This treatment provides an intensificationof the processes of nitrogen fixation and photosynthesisand stimulates growth of above-ground plant mass andrhizogenesis and leads to increased productivity ofMedicagosativa L. and improved amino acid composition of plantleaves. Phylogenetic analysis by using two differentmolecularmarkers showed that this new cyanobacterium belongs to acluster of the genus Nostoc, with the closest relative of Nostocmuscorum. Gene transfer of transposon bearing plasmidDNA has been shown for cyanobacterium Nostoc PTV. Itmakes this strain very attractive model for future genetic andphysiological experiments and biotechnological applications.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

This work was partly supported by Grant no. 14-04-00656 ofthe Russian Foundation for Basic Research. Authors devotethis study tomemory of Professor Tatjana V. Parshikova, whoinitiated this research.

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